Maleimides are versatile derivatives that find extensive use in chemical synthesis and in biological and pharmacological applications. As Michael acceptors, maleimides react readily with sulfhydryl groups to form stable thioether bonds. This reaction is extensively used with proteins and the like where both sulfhydryl and amine groups are present. At approximately neutral pH, maleimides are highly selective, with sulfhydryl groups being about 1,000 times more reactive than amine groups (Smyth et al., Biochem. J., 91, 589, 1964; Gorin et al. Arch. Biochem. Biophys. 115, 593, 1966; Partis et al., J. Protein Chem, 2, 263-277, 1983). At higher pH values of 8 or above, the reaction of maleimides with amine groups begins to significantly compete (Brewer and Riehm, Anal. Biochem. 18, 248, 1967). While best known as Michael acceptors, maleimides are also useful for their reactivity as dienophiles (Baldwin et al., Tetrahedron Lett., 32, 5877, 1991; Philp and Robertson, J. Chem. Soc., Chem. Commun., 1998, 879; Bravo et al., Heterocycles, 53, 81, 2000) and as dipolarophiles (Grigg et al., J. Chem. Soc., Perkin Trans. 1, 1988, 2693; Konopikova et al., Collect. Czech. Chem. Commun., 57, 1521, 1991; Philp and Booth, Tetrahedron Lett., 39, 6987, 1998).
Maleimide groups can be used to facilitate covalent attachment of proteins and other molecules to polymers. For example, the hydrophilic polymer “poly(ethylene glycol)”, abbreviated as “PEG”, is often used to conjugate bioactive molecules and render them soluble in aqueous media (Harris et al. “Poly(Ethylene Glycol) Chemistry and Biological Applications”, ACS Symposium Series, ACS, Washington, D.C., 1997). PEG-maleimide is an example of a reactive polymer suitable for reaction with thiol or amino groups on a biologically active molecule.
Many of the methods for preparing PEG maleimides involve connecting an activated PEG to a small linker molecule comprising a maleimide group, many of which are available commercially. There are a variety of shortcomings associated with several known PEG maleimides and methods for their production. For example, the so-called “linkerless” PEG maleimides, which have no linker group between the PEG and the maleimide group, are often prepared directly from a PEG amine using one of two methods. See U.S. Pat. No. 6,602,498. These methods, however, generally result in a relatively impure product inasmuch as a fairly significant amount of an open ring maleamic acid-containing derivative is present in the final product as will be discussed below.
In the first method disclosed in U.S. Pat. No. 6,602,498, a water soluble and non-peptidic polymer backbone is reacted with maleic anhydride to form an open ring amide carboxylic acid intermediate (a maleamic acid intermediate). The ring of the intermediate is then closed in a second step by heating the intermediate in the presence of acetic anhydride and a salt of acetic acid, such as sodium or potassium acetate, to a temperature of about 50° C. to about 140° C. for about 0.2 to about 5 hours. This two-step process is summarized in the Reaction Scheme I, provided below:

The crude maleimide-terminated, water-soluble polymer-containing composition made by this method may contain a substantial amount of the open ring maleamic acid intermediate. A major cause for the appearance of the open ring maleamic acid intermediate may lie with the heating step, especially if any acidic species is generated or is a contaminant in the acetic anhydride. Under these conditions, it is possible to isomerize the C═C bond and thus make ring closure difficult, if not impossible. As a result, it is desirable to purify the polymer product by some method, such as ion exchange chromatography, capable of removing the impurity. However, the maleimide ring system does not tolerate a chromatographic column bearing basic or nucleophilic sites, thus making purification more difficult. A second problem with this synthetic route sterns from the use of PEG amine.
Similarly, Sakanoue et al., U.S. Patent Application Publication No. 2003/0065134 A1, describes a related method except that the PEG-maleimides produced therein comprise a propylene group rather than an ethylene group between the ultimate PEG oxygen and the maleimide nitrogen. The method described in Sakanoue et al., however, suffers from the same problems as mentioned above. Further, the reference teaches that the PEG amines are generally manufactured by reduction of a nitrile group using hydrogen and a nickel catalyst, which can lead to the introduction of additional impurities due to reaction between the amine product and an imine intermediate.
In a second synthetic route described in U.S. Pat. No. 6,602,498 (the “Aqueous N-alkoxycarbonylmaleimide route”), an N-alkoxycarbonylmaleimide is reacted with a polymeric amine to form a maleimide-terminated, water-soluble polymer product. A ring-opening and ring-closing reaction occurs similar to the one described above. The reaction is conducted over a slowly increasing temperature gradient in an aqueous sodium bicarbonate buffer at a pH of about 8.5. The maleimide group, however, is not stable at those conditions and undergoes hydrolysis to maleamic acid. Therefore, two parallel reactions occur during synthesis: formation of maleimide ring and maleimide ring hydrolysis.
One approach for addressing the problem would be to stop the reaction at a time when a maximum amount of the maleimide-terminated, water-soluble polymer product is formed. While this approach appears sound in theory, it is almost an impossible task in commercial practice due to the changing reaction temperature wherein it can be difficult to reproducibly achieve the temperature gradient during consecutive manufacturing batches. For example, consecutive commercial batches of certain maleimide-terminated, water-soluble polymers were found to have maleimide purity from 65 to 80%, and maleamic acid content of about 20 to 35%. Again, chromatography is not a viable option because of the sensitivity of the maleimide group to the functional groups of the ion exchange column. Furthermore, even if it were possible to reproducibly control the temperature gradient and stop the reaction at the proper time, the approach requires close monitoring and additional equipment (e.g., thermocouples, heat jackets, and so forth), thereby adding complexity to the approach.
Other approaches for preparing maleimide-terminated, water-soluble polymers are described in International Patent Publication WO 05/056636. In one approach (labeled as “Reaction Scheme II” below) a polymer comprising a leaving group (“LG”) and a salt of an imide (shown as the potassium salt of a tricyclic amide) are reacted via nucleophilic substitution to form a polymer intermediate, which is then followed by a reverse Diels-Alder reaction to provide a maleimide functionalized polymer and furan.
Although the reaction shown above utilizes relatively simple functionalized polymers and Diels-Alder adduct reagents that react to form so-called “linkerless” maleimides (meaning the maleimide group is directly attached to the polymer), the reaction requires not commercially available reagents.
Notwithstanding the approaches described above, there remains a need to provide still other approaches for preparing maleimide-terminated, water-soluble polymers so that, for example, the approach best suited for a particular need can be used. The novel approach described herein is believed to provide, among other things, maleimide-terminated polymers in high yield and free from significant amounts of polymeric impurities, particularly significant amounts of polymer impurities that cannot be readily removed using conventional purification techniques, such as ion exchange chromatography.